Doctor of Philosophy / Genetics Interdepartmental Program - Plant Pathology / Bikram Gill / The diploid D genome species, Aegilops tauschii Coss. (2n=2x=14,DD) has provided numerous genes for resistance to diseases and insect pests that attack bread wheat (Triticum aestivum L. (2n=6x=42, AABBDD). Wheat production is currently threatened by broadly virulent races of the ‘Ug99’ lineage of wheat stem rust caused by the fungus, Puccinia graminis f.sp. tritici Pers. & Eriks. Screening of a large set of Ae. tauschii germplasm for resistance to TTKSK (Ug99) identified potentially novel sources of resistance. To expedite utilization of TTKSK resistance from Ae. tauschii, a direct hybridization approach was established that integrates gene transfer, mapping and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding was used to initiate transfer of resistance. Genetic mapping of resistance was accomplished during gene transfer through development of BC[subscript]2 mapping populations. Bulked segregant analysis of BC[subscript]2F[subscript]1 genotypes at 70 SSR loci across the D genome identified the chromosome locations of stem rust resistance genes and facilitated genetic mapping. Using this approach, TTKSK resistance from CDL4424 and TA1662 was mapped on chromosome arm 1DS flanked distally by Xwmc432 and proximally by Xwmc222 at 4.4 cM, TA10187 on 6DS linked to Xcfd49 at 1.9 cM and TA10171 on 7DS linked Xwmc827 at 0.9 cM. TTKSK resistance from additional Ae. tauschii accessions CDL4366, TA1615, TA1642, TA1693 and TA1718 has been recovered in segregating populations but await mapping. Altogether, TTKSK resistance from eight Ae. tauschii accessions has been introgressed to a hard winter wheat genetic background. Three new stem resistance genes have been tagged with molecular markers for marker assisted breeding and will provide valuable material for stem rust resistance breeding and gene pyramids for effective control of stem rust.

To improve management and control of jointed goatgrass (Aegilops cylindrica Host.) on traditional winter wheat (Triticum aestivum L.) cropland, a better understanding of the effects spring crop and wheat planting date have on weed populations and wheat yield is needed. A study of the effects of safflower (a spring crop) and wheat planting dates (early vs late) was conducted over a 2-yr period. Long term effects will be examined over a 5-yr period. The effects these treatments had on yield, weed seed contamination, jointed goatgrass population density, and soil seedbank concentration were measured. Two identical experiments were initiated, the first beginning in 1996, the second in 1997. In experiment one, initial plant counts showed similar numbers of jointed goatgrass plants in all treatments. In experiment two, initial spring plant counts showed increased numbers of jointed goatgrass in unplanted plots prior to planting safflower, and slightly reduced population densities in October-planted wheat when compared to September-planted wheat. Winter wheat yields were 25% and 35% higher in September-planted wheat than in October-planted wheat, in 1997 in experiment one, and1998 in experiment two, respectively. Crop contamination with jointed goatgrass propagules was four times higher in early vs late-planted wheat in 1997, and 36% higher in 1998. Jointed goatgrass plants in safflower were reduced 97% compared to preplan! counts in both experiments. In experiment one, 1998 fallow season plant counts showed 55% and 75% less jointed goatgrass in fallow safflower plots than in fallow plots of September- and October-planted wheat, respectively, with fallow plots of September-planted wheat having 46% less than fallow plots of October-planted wheat. Soil seed bank concentrations were highest at the 0-5 cm depth of October-planted wheat, which had nearly a 10-fold higher concentrations compared to safflower and September-planted wheat at this depth. There were no differences at depths below 5cm. This study showed the use of safflower to be a very useful management tool for reducing jointed goatgrass populations. September-planted wheat, with similar jointed goatgrass populations, yielded higher, and had less contamination and was therefore more competitive with jointed goatgrass than wheat planted in October, observed through a reduction in jointed goatgrass propagule production. Planting wheat in October, for the purpose of controlling jointed goatgrass through additional tillage, proved ineffective. Jointed goatgrass population densities were not reduced in experiment one, and only slightly reduced in experiment two. The dramatic loss of yield, associated with the later plantings, far outweighs any benefits gained by delaying wheat planting.

Doctor of Philosophy / Genetics Interdepartmental Program / Bikram S. Gill / Leaf rust, caused by fungus Puccinia triticina, is an important foliar disease of wheat worldwide. Breeding for race-nonspecific resistant cultivars is the best strategy to combat this disease. Aegilops tauschii, D genome donor of hexaploid wheat, has provided resistance to several pests and pathogens of wheat. To identify potentially new adult plant resistance (APR) genes, 371 geographically diverse Ae. tauschii accessions were evaluated in field with leaf rust (LR) composite culture of predominant races. Accessions from Afghanistan only displayed APR whereas both seedling resistance and APR were common in the Caspian Sea region. Seventeen accessions with high APR were selected for production of synthetic hexaploid wheat (SHW), using ‘TetraPrelude’ and/or ‘TetraThatcher’ as tetraploid parents. Six SHWs were produced and evaluated for APR to LR and resistance to tan spot at seedling stage. Genetic analysis and mapping of APR introgressed from accession TA2474 was investigated in recombinant inbred lines (RIL) population derived from cross between SHW, TA4161-L3 and spring wheat cultivar, ‘WL711’. Genotyping-by-sequencing approach was used to genotype the RILs. Maximum disease severity (MDS) for LR was significantly correlated among all experiments and APR to LR was highly heritable trait in this population. Nine genomic regions significantly associated with APR to LR were QLr.ksu-1AL, QLr.ksu-1BS, QLr.ksu-1BL.1, QLr.ksu-1BL.2, QLr.ksu-2DS, QLr.ksu-2DL, QLr.ksu-5AL, QLr.ksu-5DL and QLr.ksu-6BL. Association of QLr.ksu-1BL.1 with marker Xwmc44 indicated this locus could be slow-rusting APR gene, Lr46/Yr29. QTLs detected on 2DS, 2DL and 5DL were contributed by TA4161-L3 and are novel, along with QLr.ksu-5AL. Tan spot, caused by necrotrophic fungus, Pyrenophora tritici-repentis, has recently emerged as a damaging disease of wheat worldwide. To identify QTLs associated with resistance to Race 1 of P. tritici-repentis, F[subscript]2:3 population derived from cross between SHW, TA4161-L1 and winter wheat cultivar, ‘TAM105’ was used. Two major effect QTLs, QTs.ksu-1AS.1 and QTs.ksu-7AS were significantly associated with tan spot resistance and contributed by TA4161-L1. QTs.ksu-7AS is a novel QTL and explained 17% of the phenotypic variation. Novel QTLs for APR to LR and tan spot identified in SHWs add new variation for broadening the gene pool of wheat and providing resources for breeding of durable resistant cultivars.

Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / High temperature (HT) and drought are detrimental to crop productivity, but there is limited variability for these traits among wheat ([italics]Triticum aestivum[end italics] L.) cultivars. Five [italics]Aegilops[end italics] species were screened to identify HT (52 accessions) and drought (31 accessions) tolerant species/accessions and ascertaining traits associated with tolerance. Four synthetic wheats were studied to quantify independent and combined effects of HT and drought. [italics]Aegilops[end italics] species were grown at 25/19°C day/night and 18 h photoperiod. At anthesis, HT was imposed by transferring plants to growth chambers set at 36/30°C, whereas in another experiment, drought was imposed by withholding irrigation. Synthetic wheats were grown at 21/15°C day/night and 18 h photoperiod. At anthesis or 21 d after anthesis, plants were exposed to optimum condition (irrigation + 21/15°C), HT (irrigation + 36/30°C), drought (withhold irrigation + 21/15°C), and combined stress (withhold irrigation + 36/30°C). Stresses were imposed for 16 d. High temperature and drought stress significantly decreased chlorophyll, grain number, individual grain weight, and grain yield of [italics]Aegilops[end italics] species (≥ 25%). Based on a decrease in grain yield, [italics]A. speltoides[end italics] and [italics]A. geniculata[end italics] were most tolerant (~ 61% decline), and [italics]A. longissima[end italics] was highly susceptible to HT stress (84% decline). Similarly, [italics]A. geniculata[end italics] had greater tolerance to drought (48% decline) as compared to other species (≥ 73% decline). Tolerance was associated with higher grains spike [superscript]-1 and/or heavier grains. Within [italics]A. speltoides[end italics], accession TA 2348 was most tolerant to HT with 13.5% yield decline and a heat susceptibility index (HSI) 0.23. Among [italics]A. geniculata[end italics], TA 2899 and TA 1819 were moderately tolerant to HT with an HSI 0.80. TA 10437 of [italics]A. geniculata[end italics] was the most drought tolerant accession with 7% yield decline and drought susceptibility index 0.14. Irrespective of the time of stress, HT, drought, and combined stress decreased both individual grain weight and grain yield of synthetic wheats by ≥ 37%, 26%, and 50%, respectively. These studies suggest a presence of genetic variability among [italics]Aegilops[end italics] species that can be utilized in breeding wheat for HT and drought tolerance at anthesis; and combined stress of drought and high temperature on synthetic wheats are hypo-additive in nature.

Synthetic hexaploid wheats, created from a durum (Triticum durum) cross to Aegilops tauschii Coss. (McFadden and Sears, 1946), proved to be an efficient and beneficial source of new genes for common bread wheat (Triticum aestivum L). The purpose of this research was to evaluate the potential and performance of synthetic wheat in Texas. Ten elite primary synthetics from the International Maize and Wheat Improvement Center (CIMMYT), screened for desirable traits, were backcrossed to two Texas cultivars, TAM 111 and TAM 112. Populations were bulked and modified bulked to advance generations. Agronomic traits related to yield were determined on the F4 and F5 Improvement was observed in South Texas and the Blacklands, which have more disease pressure and fewer intermittent dry spells than another two locations at Chillicothe and Bushland in Texas Rolling and High Plains, respectively. Selected bulks were not superior to non-selected bulks. Head number per unit area had the highest correlation with yield and seed weight was the most heritable trait. Synthetic lines combined better with TAM 111 than TAM 112 in high yielding environments. populations across five Texas locations. Similar to crosses with spring wheat, synthetics contributed to yield through an increase in seed weight. Synthetic populations that produced higher grain yield than both TAM 111 and TAM 112 were able to maintain their large seed size and weight while improving their seed per head and head number traits. Poorer performance in environments with harsh winters could be due to a lack of winter-hardiness in the primary synthetics. This clearly demonstrates that improving yield, through utilization of common wheat by synthetic crosses, could result from selecting for larger seed per head and heads per unit area in lines driven from these populations. Introgression of new genes through synthetic backcrosses could contribute to the improvement of wheat in particular regions of Texas. Primary synthetics and recurrent parents combining for superior hybrids were identified.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Gene transfer from wild gras species to wheat is complicated by the simultaneous integration of large amounts of alien chromatin. The alien chromatin containing the target gene is inherited as a linkage block and the phenomenon is known as linkage drag. The degree of linkage drag depends on whether, and how readily, recombination occurs between the foreign and wheat chromatin. The S13 translocation line was developed by the department of Genetics, US. A cross was made between Chinese Spring and a leaf rust resistant Aegilops speltoides accession. Resistant backcross F1 was backcrossed to Chinese Spring and W84-17. S13 was selected from the backcross progeny and found to carry three rust resistance genes temporarily named LrS13, SrS13 and YrS13. Unfortunately, the resistance genes were completely linked to gametocidal (Gc) genes that were co-transferred from the wild parent. In wheat Gc genes cause reduced fertility, poor plant phenotype and hybrid necrosis. In order to use employ the rust resistance genes commercially they need to be separated from the Gc genes. At the onset of this study four putative shortened forms of the S13 translocation were provided. The four lines were identified in a homoeologous paring induction experiment (involving the test cross 04M127). This study aimed to achieve the following: (i) characterize the four recombinants with the use of molecular markers, (ii) use the knowledge gained to identify further recombinants in the 04M127 cross, (iii) identify the shortest (most useful) recombinant, and (iv) attempt to shorten the shortest recombinant form still further and thereby remove as many of the Gc genes as possible. In total, seven recombinants of the S13 translocation (04M127-1, -2, -3, -4, -7, -11 and -12; referred to as recombinant group A) were identified and characterised with microsatellite and SCAR markers. These recombinants have exchanged different amounts of foreign chromatin for wheat chromatin, but were still associated with Gc genes, showing hybrid necrosis and seed shrivelling. Some of the recombinants have lost the undesirable „brittle rachis‟ phenotype which occurs in Ae. speltoides and the S13 translocation line. In plants VII having this trait, the rachis spontaneously disarticulates after the third spikelet upon ripening of the ear. Recombinant 3 appeared to be least affected by Gc genes and was therefore used in further attempts to shorten the translocation. Recombinant 3 was crossed with wheat (W84-17) and resistant F1 (heterozygous for the translocation) were test crossed with Chinese Spring nullisomic 3A tetrasomic 3B/D plants. Thirty five resistant testcross F1 plants were identified (named recombinant group B). The resistant group B recombinants as well as nine susceptible test cross F1 (which also appeared to be recombinant) were characterised making use of microsatellites and a SCAR marker. From the results it appeared that each of the 35 resistant plants exchanged substantial amounts of Ae. speltoides chromatin for wheat chromatin. The species chromatin that remained (and which contains LrS13) is probably located either close to the 3AS telomere or within the proximal regions of 3AS and 3AL. A SCAR marker that has been developed specifically for the S13 translocation provided useful confirmation of the presence of Ae. speltoides chromatin in the 35 recombinants. If the SCAR marker proves to be tightly linked to LrS13 it may eventually be used for marker assisted selection of the resistance or it may be employed in continued attempts to reduce the amount of foreign chromatin. Seedling rust resistance tests showed that the recombinants have lost SrS13 and YrS1 during recombination. An attempt was also made to develop additional markers that specifically detect the translocation in order to further characterise the group B recombinants. Published information on Ae. speltoides specific repeated and transposon sequences were obtained and used for primer design. Unfortunately, no suitable markers could be found and the primers that were designed tended to amplify the same fragments in both the wheat and species genomes. DArT markers were also employed in an attempt to characterise the 35 group B recombinants and controls. The DArT results provided an independent verification of the results obtained with the microsatellite markers. The DArT results confirmed that the group B recombinants exchanged large amounts of species chromatin for wheat chromatin. Even though the 35 resistant group B recombinants have undergone extensive recombination they still show signs of residual Gc effects. It is believed these effects can be removed by continued backcrossing to wheat accompanied by selection against Gc symptoms. While the effects of Gc genes per se were not studied, their properties were reminiscent of those of transposable elements. Indications were that complex interactions involving the Gc genes themselves as well as genetic factors in the wheat genome may have a drastic effect on the selective survival of recombinant gametes.

The aim of this study was to analyse testcross-material that was generated during a homoeologous pairing-induction experiment. Absence of the homoeologous pairing suppressor gene, Ph1, was employed to induce meiotic pairing between the Lr59 translocation (Aegilops peregrina) and 1AL of normal wheat. The study aimed to characterize the test-cross plants derived from this experiment and to identify recombinants which retained the least amount of species chromatin but which still contained the Lr59 gene. The test-cross F1 population, 07M5 (total 635 plants), was screened for Lr59 resistance by inoculating seedlings with the leaf rust pathotype, UVPrt8. The 168 resistant plants were characterized with molecular markers in order to identify recombinants. The data were used to construct a physical map which showed the relative sizes of the recombinants and which could be used to identify those recombinants which contained the least amount of residual species chromatin. Microsatellite (Xcfa2219, Xbarc83 and Xgwm164) and SCAR (S15T3) analysis was used for the initial identification of recombinants. The results showed that 152 of the 168 resistant plants were recombinants for the four loci; that eight of the remaining 16 plants represented non-recombinant, wild species-types and that the last eight plants represented the wheat parental-types which were resistant (and thus, also recombinants). This extremely high recombination frequency can largely be attributed to strong segregation distortion that was evident in the cross. It is also possible that the translocation segment could derive from the S genome rather than the U genome of Ae. peregrina. The S genome is closer related to the wheat genomes than the U genome and may be more prone to recombination. With the use of the microsatellite and SCAR data, a physical map was constructed which showed the relative location of the Lr59 gene on the translocation. It appeared that the eight shortest recombinants retained terminal species chromatin. In an attempt to characterize the eight recombinants, additional marker loci had to be identified within that region. RAPD, iv AFLP and DArT markers were investigated for this purpose. RAPD analyses did not produce any useful markers. AFLP and DArT analyses did identify useful markers with which the eight recombinants could be screened. The data showed which recombinants probably retained the least amount of species chromatin. Seeing that AFLP and DArT markers are anonymous and that the distances between marker loci are unknown, it is not possible to say which recombinant is the shortest and consequently it will be nessecary to also evaluate the group of eight recombinants agronomically in order to identify the most useful ones. The results showed that multiple cross-overs apparently occured on both sides of Lr59. Multiple cross-overs are higly unlikely in material of this nature, therefore it was speculated that the observation resulted from incomplete synteny between the telomeric areas of the translocation and 1AL. A structural difference between the two chromosome regions might have given rise to abnormal meiotic pairing structures and thus unexpected gamete genotypes. Each of the eight recombinants did express one or more of the Ae. peregrina derived AFLP loci which can in future be verified for use as a marker for marker assisted selection. The study succeeded in identifying a number of potentially useful recombinants which contain the Lr59 resistance. It would, however, be risky to select only one of the shortest recombinants for further development on the basis of the present knowledge as some recombinants may contain genetic abnormalities which resulted from reduced synteny in the Lr59 region. It would therefore be wise to further evaluate all eight recombinants before the best one is selected for agronomic use.

Thesis (MSc (Genetics))--Stellenbosch University, 2008.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / The wild relatives of crop plants are sources of useful genes, but such genes when transferred to agricultural crops are often associated with deleterious traits. Because most of the recombination and the disease resistance genes are localized towards the ends of wheat chromosomes, cryptic terminal alien segments, carrying rust resistance genes, were transferred from Aegilops geniculata (UgMg) and Ae. triuncialis (UtCt) into common wheat without the usual linkage drag. The alien segment with the leaf rust and stripe rust resistance genes Lr57 and Yr40 in translocation T5DL•5DS-5MgS(0.95) was found to be less than 3.3 cM in genetic length and spans less than four overlapping BAC/PAC clones of the syntenic rice chromosome arm 12L. The alien segment with leaf rust resistance gene Lr58, transferred from Ae. triuncialis, was found to be less than 5% of the chromosome arm 2BL of wheat in T2BS•2BL-2tL(0.95), further suggesting that it is feasible to transfer small alien segments with disease resistance genes. Resistance genes Lr57, Yr40 and Lr58 were transferred to Kansas hard red winter wheat cultivars by backcrossing and marker assisted selection. Tillering, a key component of grain yield, and seed color which influences seed dormancy and pre-harvest sprouting in wheat, are agronomically important domestication traits in wheat. A tiller inhibition mutant with monoculm phenotype was isolated and the mutated gene (tin3) was mapped on the distal region of chromosome arm 3AmL of T. monococcum. As a first step towards isolating candidate gene(s), the tin3 and the seed color gene (R-A1) of chromosome 3A were mapped in relation to physically mapped ESTs and STS markers developed based on synteny with rice. Physically mapped wheat ESTs provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 and R-A1 region. Comparative genomic analysis of the tin3 and R-A1 genomic regions with the corresponding region in rice localized the tin3 gene to a 324 kb region spanned by two overlapping BACs and the R-A1 gene was mapped to a single BAC of the colinear rice chromosome arm 1L.

The objective of this study was to identify DNA markers for each of six added C-genome chromosomes in Triticum aestivum L. cv. 'Alceso'-Aegilops caudata L. addition lines using the randomly amplified polymorphic DNA (RAPD) technique. DNA from Ae. caudata, T. aestivum, amphiploid of T. aestivum X Ae. caudata, and six disomic addition lines of wheat having a pair of Ae. caudata chromosomes was used as the template for the amplification of RAPD markers with a total of 58 random 10-mer oligonucleotide primers. Two primers, OPC-08 and OPJ-16, produced one intense band each from the amphiploid of T. aestivum X Ae. caudata and Ae. caudata, which was absent in all six addition lines. Each of these two primers produced a chromosome marker that could be tentatively located to the chromosome CA of Ae. caudata. OPJ-02, OPD-12, OPD-02, OPJ-12, OPD-20, and OPJ-14 produced a marker each for CB, CC, CD, CE, CF, and CG, respectively. OPJ-09 produced C-genome chromosome-specific RAPD markers. Also, OPC-05 and OPJ-19 produced RAPDs from both wheat and Ae. caudata genomes.